Mechanism of Acidification of the trans-Golgi Network (TGN)

Abstract

Sorting of secretory cargo and retrieval of components of the biosynthetic pathway occur at the transGolgi network (TGN). The pH within the TGN is thought to be an important determinant of these functions. However, studies of the magnitude and regulation of the pH of the TGN have been hampered by the lack of appropriate detection methods. This report describes a noninvasive strategy to measure the luminal pH of the TGN in intact cells. We took advantage of endogenous cellular mechanisms for the specific retrieval of TGN resident proteins, such as TGN38 and furin, that transit briefly to the plasma membrane. Cells were transfected with chimeric constructs that contained the internalization and retrieval signals of TGN resident proteins, and a luminal (extracellular) epitope (CD25). Like TGN38 and furin, the chimeras were shown by fluorescence microscopy to accumulate within the TGN. During their transient exposure at the cell surface, the chimeras were labeled with extracellular anti-CD25 antibodies conjugated with a pH-sensitive fluorophore. Subsequent endocytosis and retrograde transport resulted in preferential labeling of the TGN with the pH-sensitive probe. Continuous, quantitative measurements of the pH of the TGN were obtained by ratio fluorescence imaging. The resting pH, calibrated using either ionophores or the "null point" technique, averaged 5.95 in Chinese hamster ovary cells and 5.91 in HeLa cells. The acidification was dissipated upon addition of concanamycin, a selective blocker of vacuolar-type ATPases. The counterion conductance was found to be much greater than the rate of H ؉ pumping at the steady state, suggesting that the acidification is not limited by an electrogenic potential. Both Cl ؊ and K ؉ were found to contribute to the overall counterion permeability of the TGN. No evidence was found for the presence of active Na ؉ /H ؉ or Ca 2؉ /H ؉ exchangers on the TGN membrane. In conclusion, selective retrieval of recombinant proteins can be exploited to target ion-sensitive fluorescent probes to specific organelles. The technique provides real-time, noninvasive, and quantitative determinations of the pH, allowing the study of pH regulation within the TGN in intact cells. The acidic pH of the TGN reflects active H ؉ pumping into an organelle with a low intrinsic H ؉ permeability and a high conductance to monovalent ions.The pH prevailing in the lumen of endomembrane compartments is an important determinant of their function. The concentration of H ϩ dictates the dissociation and degradation of internalized ligands (1) and modulates the processing, transport, and sorting of secreted proteins (2-4). Delivery of pHsensitive probes by pinocytosis or receptor-mediated internalization has facilitated the study of the pH along the endocytic pathway, revealing that the acidity of the lumen increases progressively, from a pH of 6.5 to 6.0 in early and recycling endosomes to 4.5 in lysosomes (5). Conversely, the pH inside subcompartments of the secretory pathways is thought to be highest in the endoplas...

N- and C-terminal Domains Direct Cell Type-specific Sorting of Chromogranin A to Secretory Granules

“…Ref. 30), and this aggregation has repeatedly been suggested to play a role in sorting of regulated secretory proteins. Interestingly, aggregation is neither necessary nor sufficient for sorting in PC12 cells (12,16).…”

Molecular Cloning and Characterization of a Novel (Na+,K+)/H+ Exchanger Localized to the trans-Golgi Network

“…The location of the TGN was defined by transiently transfecting the cells with an ex- pression vector containing the chimeric gene CD25-TGN38. This chimera, which is composed of the extracellular domain of the ␣-chain of the interleukin-2 receptor linked to the transmembrane and cytosolic domains of TGN38, has been shown to accumulate in the TGN and can be readily labeled with commercially available FITC-conjugated CD25 antibodies (15,22). As shown in Fig.…”

“…It has been suggested that because the pump is electrogenic, its activity could be influenced by the membrane potential and by the availability of permeant counterions such as chloride and potassium (14). However, in the case of the Golgi complex, the endogenous counterion conductances were found to exceed the rate of H ϩ pumping at the steady state, implying that the electrical potential across the membrane is negligible and therefore not a defining factor in setting organellar pH (15)(16)(17). In addition, despite extensive work, differential control of V-ATPase activity by hormones or other factors has not been found along the endo-or exocytic pathways.…”

“…In addition, despite extensive work, differential control of V-ATPase activity by hormones or other factors has not been found along the endo-or exocytic pathways. Rather, the luminal [H ϩ ] is thought to be regulated by a complex interplay between the V-ATPase and unidentified leak pathways for protons, based on the rapid dissipation of the transmembrane proton chemical gradient (⌬pH) observed after inhibiting the V-ATPase with macrolide antibiotics (15,16,18). A component of this H ϩ leak in the Golgi complex was recently identified as a Zn 2ϩ -inhibitable H ϩ conductance (17) but could not fully account for H ϩ turnover.…”

Cellubrevin-targeted Fluorescence Uncovers Heterogeneity in the Recycling Endosomes

“…Preincubation with unlabeled F(ab) significantly reduced subsequent uptake of FITC-F(ab), demonstrating that Cb indeed cycled between endosomes and the plasma membrane. The "targeted fluorescence" strategy has also been useful for examining pH of the TGN and may be a general strategy for studying organelles containing proteins that cycle between the cytosol and the plasma membrane (33,35).…”